High concentrations of saline in soils inhibit plant growth and further inhibit agricultural productivity. Modem agricultural practices including irrigation are known to increase salt concentrations when the available irrigation water evaporates, leaving dissolved salts behind. In areas containing salty soils such as Southern California, Arizona, New Mexico and Texas, it has become particularly important to develop salt tolerant cultivars of agronomically important crops. Salty soils decrease the rate at which water will enter the roots due to the osmotic pressure of the solution. If the salt concentrations are sufficiently high, water will actually be withdrawn from the plant roots, eventually leading to plant death.
Thus, development of salt tolerant cultivars is required to offset effects of irrigation, and for utilization of marginal agricultural areas, and use saltier irrigation water.
Traditional plant breeding methods require long term selection and testing to identify new cultivars. Thus far, these methods have not yielded crop plants cultivars with substantially improved salt tolerance.
Excessive sodium ions (Na+) are toxic to plants because of adverse effects on cellular metabolism and ion homeostasis. Sodium ions in saline soils are toxic to plants due to their adverse effects on K+/Na+ homeostasis, cytosolic enzyme activities, photosynthesis and metabolism (Niu, et al. (1995) Plant Physiol. 109:735-742; Jacoby B. (1999) in Handbook of Plant and Crop Stress, ed. Pessarakli M (Marcel Dekker, NY), pp. 97-123)). Niu et al. (1995), supra, report three mechanisms that prevent accumulation of Na+ in the cytoplasm: restricted Na+ influx; active Na+ efflux and compartmentalization of Na+ in the vacuole. One example of restricted Na+ influx includes the wheat low affinity cation transporter LCT1 which may mediate Na+ influx into plant cells (Schachtman et al. (1997) PNAS 94:11079-84)). Another restriction on Na+ influx includes wheat high-affinity K+ transporter HKT1 which functions as a Na+-K+ co-transporter, which confers low-affinity Na+ uptake at toxic Na+ concentrations (Rubio et al. (1995) Science 270:1660-1663). Also, non-selective cation channels have been found to play important roles in mediating Na+ entry into plants (Amtmann et al. (1998) Adv. Bot. Res. 29:76-112)).
An example of Na+ compartmentalization in the vacuole includes the Arabidopsis thaliana ATNHX1 gene which encodes a tonoplast Na+/H+ antiporter (Gaxiola et al. (1999) PNAS 96:1480-85)).
Active Na+ efflux transporters have been found in fungi. In the yeast Saccharomyces cerevisiae, plasma membrane Na+-ATPases play a predominant role in Na+ efflux and salt tolerance (Haro et al. (1991) FEBS Lett. 291:189-191)). In the fungus Schizosaccharomyces pombe, Na+/H+ antiporters are more important for Na+ efflux and salt tolerance (Jia et al. (1992) EMBO J 11:1631-40)).
Arabidopsis is a glycophyte that is not very salt tolerant, but can adapt to elevated salt concentrations. Several Arabidopsis SOS mutants defective in salt tolerance have been characterized (Wu et al. (1996) Plant Cell 8:617-27; Liu et al. (1997) PNAS 94:14960-64; and Zhu et al. (1998) Plant Cell 10:1181-1191)). The SOS mutants are hypersensitive to high external Na+ or Li+ and also are unable to grow under very low external K+ concentrations (Zhu et al., (1998), supra)). The SOS mutants are defined as three loci, SOS1, SOS2 and SOS3 (Zhu et al. (1998), supra)). The SOS3 gene has homology to animal neuronal calcium sensors and the yeast calcineurin B subunit (Liu et al. (1998) Science 280:1943-45)). In yeast, mutations in calcineurin B lead to increased sensitivity of yeast cells to Na+ and Li+ concentration (Mendoza et al. (1994) J. Biol. Chem. 269:8792-96)). The SOS2 gene encodes a serine/threonine type protein kinase (Liu et al. (2000), PNAS, in press). Halfter et al. (2000) PNAS, in press, report that the SOS2 protein physically interacts with and is activated by SOS3, suggesting an SOS2/SOS3 regulatory pathway for Na+ and K+ homeostasis and salt tolerance in plants.
The SOS1 mutant is more sensitive to Na+ and Li+ stresses than the SOS2 and SOS3 mutant plants (Zhu et al. (1998), supra)). Double mutant analysis indicates that SOS1 functions in the same pathway as SOS2 and SOS3 (Liu et al. (1997), supra; and Zhu et al. (1998), supra)). The SOS1 protein may be a target for regulation by the SOS3/SOS2 pathway. The SOS1 gene has been cloned from Arabidopsis thaliana (U.S. Pat. No. 6,727,408). The SOS1 protein has Na+/H+ transporter activity and homology to Na+/H+ antiporters from bacteria and fungi. SOS1 transcript is up-regulated by NaCl stress. The SOS2 mutation abolishes SOS1 up-regulation in the shoot. In the SOS3 mutant, no SOS1 up-regulation is found in the shoot or root. SOS1 gene expression appears therefore to be regulated under NaCl stress by the SOS3/SOS2 regulatory pathway. (U.S. Pat. No. 6,727,408).
The salt cress, or Thellungiella halophila, can withstand dramatic salinity up to 500 mM NaCl and grow in salt far in excess of the capability of Arabidopsis. Salt cress has been suggested to be a favorable model for further study of salt tolerance because it has desirable life history traits (small size, short life cycle, self-pollination and high seed number), and favorable genetic traits (self-fertilization, a small genome, efficient transformation and mutagenesis). The salt cress genome is less than twice the size of Arabidopsis. EST analysis of hundreds of salt cress clones indicates 90-95% identities between Arabidopsis and salt cress cDNAs and amino acid sequences (Bressan et al. (2001) Plant Physiol. 127:1354).
A full-length cDNA microarray of Arabidopsis containing thousands of cDNAs including SOS1, has been used for expression profiling of salt cress genes. It was found that 6 genes were strongly induced in salt cress in response to high salinity stress, whereas 40 genes were identified as salt stress-inducible in Arabidopsis. The expression profiles of genes highly expressed in salt cress under normal growth conditions (not high salinity) were reported to resemble those Arabidopsis genes induced under abiotic stress (such as high salinity). It was suggested that salt cress constitutively overexpresses a large number of genes, including SOS1, even under unstressed conditions (Taji et al. (July 2004) Plant Physiol. 135:1697). Although Taji et al. report that an SOS1 gene is expressed in salt cress, they do not provide the sequence or report whether the salt cress SOS1 gene confers salt tolerance to salt cress.
Inan et al. (July 2004) Plant Physiol. 135:1718, report that the salt cress SOS1 gene is 84% homologous to the Arabidopsis SOS1 gene. This level of homology is less than the 90-95% homology observed between Arabidopsis and salt cress housekeeping genes.